The present invention relates to "activated" immature astrocytes and the method of utilizing the activated immature astrocytes in injectable form or on nitrocellulose polymer as a means for reducing secondary necrosis and glial scar formation in lesions in the brain and spinal cord as well as promoting directed blood vessel and axon growth and/or regeneration. The activated immature astrocytes and pharmaceutical compositions comprising same, may be used to treat disorders of the nervous system resulting from accidents or diseases which have in some way damaged the nerve tissue.
In the central nervous system the chief non-nervous cells are the glial cell types. These vary in numbers and type from one part of the nervous system to another, but the two basic classes can be distinguished by their size and embryonic origin, namely the marcroglia, i.e. which are relatively large cells derived from the neural plate, and the smaller microglia which stem from the mesodermal tissues surrounding the nervous system.
The microglia comprise two cell types, the astrocytes (astroglial cells) and the oligodendrocytes (oligodendroglial cells). The present invention is directed to the use of the former, i.e. astroglial cells, in their immature "activated" state, to reduce both secondary cell death and glial scar formation and promote axon regeneration and blood vessel growth.
Astrocytes possess small cell bodies (the nucleus is about 8 mm in diameter in man) with ramifying dendrite-like extensions. The cytoplasmic processes of astrocytes carry fine, foliate extensions which partly engulf and separate neurons and their neurites, and often end in plate-like expansions on blood vessels, ependyma and on the pial surface of the central nervous system.
The functions of astrocytes are numerous. They act mechanically as a supporting component of the nervous system. Their microfilaments, microtubules, and surface contact zones fit them for this task. They also act defensively by phagocytosing foreign material or cell debris. They can function as antigen presenting cells to macrophages and can provide a means of limited repair by forming glial scar tissue or filling the gaps left by degenerated neurons. In addition, they have essential metabolic functions in regulating the biochemical environment of neurons, providing nutrients, and regulating acid-base levels, etc.
Moreover, the astrocytes, which are able to divide in immature and mature animals, pass after mitosis through a series of structural transformations depending on their state of maturity. In areas of brain injury in young or old animals they proliferate (gliosis) to produce neural support. In a penetrating injury to the central nervous system (CNS) of adult mammals, severe tissue damage and secondary necrosis occurs in the region surrounding the wound. The degenerating effects caused by the injury are believed to generate a response in the surviving glial cells adjacent to the site of the injury (Reier et al., The Astrocytic Scar As an Impediment to Regeneration in the Central Nervous System, Spinal Cord Reconstruction, Raven Press, N.Y. pp. 163-195, 1983). The astrocyte response consists of a slight mitotic increase, an increase in size (hypertrophy), and a concomitant increase in quantity of intermediate filaments (Mathewson, et al., Observations on the Astrocyte Response to a Cerebral Stab Wound in Adult Rats, Brain Res., 327: 61-69, 1985). Together with invading monocytes, the astrocytes act as phagocytes to clear debris within the wound cavity. (Schelper, et al., Monocytes Become Macrophages: They do not become Microglia: A Light and Electron Microscopic Autoradiographic Study Using 125-Iododeoxyuridine, J. Neuropath., and Exper. Neurol., 45: 1-19, 1986). When the injury disrupts the plial lining of the brain, fibroblasts migrate into the wound cavity and multiple layers of basal lamina form over the astrocyte surface (Bernstein, et al., Astrocytes Secret Basal Lamina after Hemisection of the Rat Spinal Cord, Brain Res., 327: 135-141, 1985). The fibroblasts also produce collagen, which forms dense bundles within the surrounding extracellular spaces several weeks after injury. Thus, in adults the astrocytes, together with other cellular elements, form dense interwoven scars which fill the space vacated by the dead or dying cells in the injury area. Although the scar may help save the organism it also blocks axonal regeneration and the individual is left with an irreversible functional deficit or epileptic focus depending on the site of the lesion.
One embodiment of the present invention relates to the use of "activated" immature astrocytes to reduce the glial scar formation produced as described above. Previous studies by the inventors and others indicated that penetrating lesions in the central nervous system (CNS) of neonatal mammals rarely resulted in the formation of glial scars similar to those observed in adults and that the production of typical adult glial scars after injury increased after the first two postnatal weeks in rodents. (Barrett, et al., Differences Between Adult and Neonatal Rats in their Astroglial Response to Spinal Injury, Exp. Neurol., 84: 374-385, 1948; and Smith, et al., Changing Role of Forebrain Astrocytes During Development, Regenerative Failure, and Induced Regeneration Upon Transplantation, J. Comp. Neurol. 251: 23--43, 1986).
Moreover, the inventors have recently discovered in young animals implanted in their cerebral cortices with cellulose filters before postnatal day 8 (P8), that astrocytes did not produce a scar around the implant but instead sent many processes into the pores of the filter "suturing" it into the CNS. In contrast, astrocytes in older mice (implanted on or later than postnatal day 14)failed to incorporate the filter within the brain and, instead, produced a glial-mesenchymal scar around the filter which, in turn, did not support axon growth. The age related changes in the CNS response to wounding and the incorporation of the implant indicated the presence of a critical period wherein "activated" immature astrocytes (postnatal day 8 or less) repressed scar formation and post-critical astrocytes (postnatal day 14 or greater) produced glial scars. One embodiment of the present invention is directed to this discovery of the "cell suturing" phenomenon, wherein activated immature astrocytes are transplanted on polymer or as an injected suspension from a critical period animal to a post-critical period animal to reduce glial scar formation.
An additional embodiment of the present invention relates to a method for utilizing activated immature astrocytes as a means for promoting directed axon regeneration. Previous studies have demonstrated that CNS axons have the potential to grow long distances through peripheral nerve grafts (Friedman, et al., Injured Neurons in the Olfactory Bulb of the Adult Rat Grow Axons along Grafts of Peripheral Nerve, J. Neurosci. 5: 1616-1625, 1985) or Schwaan cell bridges (Kromer, et al., Transplantation of Schwaan Cell Cultures Promote Axonal Regeneration in the Adult Mammalian Brain, Proc. Natl. Acad. Sci. 82: 6330-6334. 1985). However, the studies with peripheral nerve elements indicated that regenerating nerve fibers could only extend a short distance upon reentry into the CNS most likely due to the formation of scars at the ends of the graft. Thus, although the injured adult CNS is potentially capable of a considerable amount of regeneration, sprouting is usually abortive and the axons fail to reinnervate their appropriate targets.
In addition, studies by the inventors indicated that developing axons are guided by oriented "highways of astroglial tissues". (Silver, et al., Studies on the Development of the Eye Cup and Optic Nerve in Normal Mice and Mutants with Congenital Optic Nerve Aplasia, Dev. Biol. 68: 175-190, 1979; Silver et al., Axonal Guidance During Development of the Great Cerebral Commissures: Descriptive and Experimental Studies, in Vivo, on the Role of Preformed Glial Pathways, J. Comp. Neurol., 210: 10-29, 1982). In lesioned areas of the embryonic brain that lack guidance pathways the forming axon tracts gather into massive neuromas. Perhaps the most dramatic demonstration of such an axonal disorder was shown in rats having surgically induced acallosal malformations. When a glial bridge is lesioned embryonically all or most of the fibers of the largest axonal pathway in the mammalian brain, the corpus callosum, failed to cross into opposite cerebral hemispheres. These axons did not die. Instead, as the fibers arrived on schedule at the hemisphere midline, they gathered into massive, paired neuromas (Probst's bundles) adjacent to the longitudinal cerebral fissure.
Further, the inventors have shown that in early postnatal lesion-induced acallosal animals, that an untreated, properly shaped nitrocellulose (Millipore) filter, placed adjacent to the neuromas and spanning the lesioned cerebral midline, could support the migration of immature glia (Silver, et al., Postnatally Induced Formation of the Corpus Callosum in Acallosal Mice on Glia-Coated Cellulose Bridges, Science, 220: 1067-1069, 1983). The glia attached to the surface of the filter to produce a cellular scaffold, which in turn provided a terrain suitable for the ectopic axons in the neuromas to traverse the midline to reform the corpus callosum.
However, the inventors only recently determined that a "critical period" existed for the formation of induced callosal axon growth to occur. The inventors noted that the stellate-shaped, GFAP-positive "activated" immature astrocytes migrated and attached to the implant by inserting a foot of their cytoplasmic processes into the pores of the filter implant. This form of gliotic response only occurred in animals younger than the eighth postnataly day (P8) and established an axon growth promoting substratum within 24-48 hours after implantation. Hence, an additional embodiment of the present invention is directed to the use of only "activated" immature (i.e. postnatal day 8 or less) astrocytes for promoting axonal regeneration.
Similarly, a further embodiment of the present invention concerns the use of "activated" immature astrocytes in injectable form or on nitrocellulose implants to promote directed axon regeneration and reduce glial scar formation in damaged spinal axons of the central nervous system. The inventors and others have demonstrated after repeatedly crushing or cutting the dorsal roots near their entrance point in the spinal cord, that the peripheral sensory fibers are regenerated only as far as the dorsal root entry zone (DREZ) of the spinal cord but no further. The problem at the DREZ is analogous to the failure of axon regeneration throughout the remainder of the CNS. Although the distance needed to reconnect the regenerating sensory fibers with their denervated dendrites in the dorsal horn of the spinal cord is relatively short (i.e. only fractions of a millimeter in the adult rat), this scant distance is normally never breached by regenerating sensory fibers in adult animals.
In order to span the gap between the sensory fibers and the denervated dendrites in the dorsal horn of the spinal cord, the inventors have developed a process for bridging the root-cord interface with a newly designed "pennant-shaped" nitrocellulose polymer (8 um pore size) implant coated with activated immature astrocytes. The results of the process indicate that the combination of the activated immature astrocytes plus the specially designed polymer implant represses scar formation locally in the cord dorsal root entry zone and stimulates axons and blood vessels to enter the CNS along the bridge surface. In addition, animals with dorsal root lesions and inserted activated immature astrocyte coated implants, exhibited remarkable functional recovery of many basic sensory-motor behaviors. Hence, a further embodiment of the present invention is directed to the use of a specially designed nitrocellulose implant coated with activated immature astrocytes to promote directed axon regeneration and repress scar formation of damaged spinal axons of the central nervous system.